Coagulation or clotting of blood involves a highly complex process of a series of interactions between several enzymes and substrates. At its simplest level it involves thromboplastin (or tissue factor), an enzyme produced by damaged tissue which converts a protein in the plasma, prothrombin, into thrombin. Thrombin then converts another plasma protein, fibrinogen, into fibrin, a filamentous protein which forms a mesh work in which red and white blood cells and platlets become entangled.
This classic model provides an oversimplified version of the clotting process as numerous other enzymes are involved. For example Factor III, tissue factor; Factor IV, calcium ions; Factor V, an unstable protein substance proaccelerin; Factor VII, proconvertin or serum prothrombin conversion accelerator; Factor VIII antihemophilic factor; Factor IX Christmas factor; Factor X Stuart-Prower factor; Factor XI plasma thromboplastin anticedent (PTA); Factor XII Hageman or glass factor; Factor XIII fibrin stabilizing factor (FSF); and prekallikrein, also called Fletcher Factor just to name a few. This initial provisional matrix of fibrin and fibronectin produced through activation of these hemostatic pathways constitutes the first phase (Inflammatory Phase) of a cutaneous wound repair. Traditionally neutrophils infiltrate the provisional matrix followed by monocytes and macrophages.
The next stage includes new tissue reformation. During this stage re-epithelialization is produced by migration and proliferation of basal keratinocytes over the provisional matrix. Granulation tissue is formed through macrophage accumulation, fibroblast ingrowth, and angiogenosis. Proliferation of myofibroblasts leads to contraction of the wound.
The final phase includes matrix remodeling in which down-regulation of fibroblast proliferation is necessary for wound maturation and prevention of hypertrophic scar formation. During this phase, fibronectin is removed from the matrix and replaced by proteoglycans and fibrous bundles of type I collagen.
Several physiological mediators have been proposed to stimulate various aspects of the three phases of cutaneous wound repair. Among the proposed mediators are fibrin, components of the complement pathway, platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), transforming growth factors .alpha. and .beta., fibroblast growth factors, and epidermal growth factor (EGF).
More recently, thrombin has become recognized as a potential stimulator of keratinocytes, fibroblasts, macrophages, and endothelial cells during wound healing. These effects of thrombin appear to be mediated partly through stimulation of a proteolytically-activated thrombin receptor, and partly through other unidentified receptors. Thrombomodulin inhibits thrombin-mediated stimulation through multiple mechanisms. First, thrombomodulin directly inhibits stimulation through the proteolytically-activated receptor by competing for thrombin binding. Second, thrombomodulin stimulates the protein C anticoagulant pathway, which decreases thrombin production, thereby indirectly inhibiting thrombin stimulation through all receptors.
More specifically thrombomodulin forms an approximately one-to-one stoichiometric complex with thrombin. In this complex form, thrombin fails to react with its natural substrates including fibrinogen, Factor V and platelets. Additionally this complex when formed, enhances the ability of thrombin to activate protein C more than 1,000 fold. Activated protein C functions as a potent natural anticoagulant by inactivating coagulation factors Va and VIIIa. Thus thrombomodulin functions to convert thrombin from a procoagulant protease to an anticoagulant.
Thrombomodulin was initially identified as an endothelial cell protein and immunohistochemical studies have demonstrated it to be present on endothelial cells throughout the vasculature. Constitutive expression of thrombomodulin on the luminal surface of blood vessels localizes coagulation to sites of vascular injury.
Thrombomodulin has been proposed to be a specific marker for cells of endothelial origin. However thrombomodulin is also synthesized in varying amounts by other types of cells including syncytiotrophoblast platelets megakaryocytes, monocytes, neutrophils, and synovial lining cells.
The association of cutaneous thrombosis with severe protein C deficiency suggests that the thrombomodulin/protein C pathway is particularly important in skin. Thrombomodulin is known to be expressed by endothelial cells of dermal vessels. Attempts to identify thrombomodulin as present in human epidermis, a nonvascular tissue, however, have been inconclusive to date.
Thrombomodulin was initially identified and purified in 1981 as an endothelial cell co-factor for activation of the anticoagulant protein C. The cDNA for human thrombomodulin was subsequently cloned by three groups (in Saint Louis, Boston, and Japan) in 1987. Human thrombomodulin is currently being developed for therapeutic use as an anticoagulant.
This invention relates to the discovery that thrombomodulin regulates thrombin function not only during primary hemostasis but also during the processes of wound healing and tissue repair. Evidence exists that thrombin is involved in angiogenesis (by stimulating vascular endothelial cell proliferation), granulation tissue formation (by stimulating macrophages and fibroblasts), and re-epithelialization (by activating basal layer keratinocytes). Thus disordered wound healing (such as excessive scar formation or chronic ulceration) could be controlled by regulating thrombin activity by thrombomodulin. A similar process may occur in non-cutaneous wounds such as those in the gastrointestinal or urinary tracts.
It is an object of the present invention to provide a method of enhancing wound healing by regulating thrombin activity through introduction of thrombomodulin to sites of wound repair.
It is yet another object of the present invention to further study and disclose the role of thrombomodulin in wound healing and epithelial cell differentiation.
Yet another object is to provide agents to help prevent scar formation from cutaneous injuries.
Yet another object is to provide a method for preventing excess scarring by use of thrombomodulin.